Enhanced isotachophoresis assays using additives with spatial gradients

ABSTRACT

Techniques for enhanced isotachophoresis assays using additives with spatial gradients include forming a concentration gradient of an additive along a channel from an input port to an output port. The channel is used for isotachophoresis with ions of a leading electrolyte having a first mobility greater than a mobility of an analyte, and ions of a trailing electrolyte having a second mobility less than the mobility of the analyte. The additive is different from both the leading electrolyte and the trailing electrolyte; and the additive has a third mobility that assures the analyte will encounter the additive. The method further comprises introducing a mixture of the trailing electrolyte and a sample including the analyte. The method further comprises applying an electric field to the channel; and, measuring the analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit as a Continuation of application Ser.No. 13/252,138, filed Oct. 3, 2011, the entire contents of which arehereby incorporated by reference as if fully set forth herein, under 35U.S.C. § 120; which claims benefit of Provisional Appln. 61/388,921,filed Oct. 1, 2010, the entire contents of which are hereby incorporatedby reference as if fully set forth herein, under 35 U.S.C. § 119(e).

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with Government support under contractN66001-09-C-2082 awarded by the Defense Advanced Research ProjectsAgency and under contract TR000093 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Isotachophoresis (ITP) separates charged molecules (ions) in a samplebased on their different effective mobility. Effective mobility is theproportionality constant between observable drift velocity of an ion andapplied electric field. It is a function of ion shape, size, and thedegree of ionization.

ITP uses a leading electrolyte (LE) which contains a relatively highmobility ion, and a trailing electrolyte (TE) with a relatively lowmobility ion. The TE and LE ions are chosen to have effective mobilitiesrespectively lower and higher than target analyte ions of interest. Thatis, the effective mobility of analyte ions is higher than that of the TEand lower than that of the LE. These target analytes have the same signof charge as the LE and TE ions (i.e., a co-ion). An applied electricfield causes LE ions to move away from TE ions and TE ions to trailbehind. A moving interface forms between the adjacent and contiguous TEand LE zones. This creates a region of electric field gradient(typically from the low electric field of the LE to the high electricfield of the TE). Analyte ions in the TE overtake TE ions but cannotovertake LE ions and accumulate (“focus”) at the interface between TEand LE. Alternately, target ions in the LE are overtaken by the LE ions;and also accumulate at interface.

SUMMARY OF THE INVENTION

Techniques are provided for enhanced ITP assays using additives withspatial gradients.

According to a first set of embodiments, a method includes forming aconcentration gradient of an additive along a channel from an input portto an output port. The channel is used for isotachophoresis with ions ofa leading electrolyte having a first effective mobility magnitudegreater than a mobility of an analyte, and ions of a trailingelectrolyte having a second effective mobility magnitude less than themobility of the analyte. The additive is different from both the leadingelectrolyte and the trailing electrolyte; and the additive has a thirdeffective mobility that assures the analyte will encounter the additive.The method further comprises contacting a sample including the analyteto the leading electrolyte and contacting the trailing electrolyte tothe sample. The method further comprises applying an electric field tothe channel; and, measuring the analyte.

An additive has a mobility that assures the analyte will encounter theadditive when: the additive has a (signed) effective mobility greaterthan that of the TE in the case of anionic ITP; or, the additive has a(signed) effective mobility less than that of TE in the case of cationicITP. As used herein, measuring the analyte includes detecting,quantifying, collecting, extracting and otherwise using the analyte.

According to various other sets of embodiments, an apparatus comprisesmeans to perform each step of at least one of the above methods.

Still other aspects, features, and advantages of the invention arereadily apparent from the following detailed description, simply byillustrating a number of particular embodiments and implementations,including the best mode contemplated for carrying out the invention. Theinvention is also capable of other and different embodiments, and itsseveral details can be modified in various obvious respects, all withoutdeparting from the spirit and scope of the invention. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements and in which:

FIG. 1A is a diagram that illustrates an example isotachophoresischannel with a spatial gradient of an additive prior to application ofan external electric field, according to an embodiment;

FIG. 1B is a diagram that illustrates an example isotachophoresischannel with a spatial gradient of an additive after application of anexternal electric field, according to an embodiment;

FIG. 1C is a graph that illustrates an example electric field in thechannel at a time that corresponds to FIG. 1B;

FIG. 2A is a block diagram that illustrates an example isotachophoresischannel formed in a glass substrate, according to an embodiment;

FIG. 2B is a block diagram that illustrates an example apparatus formeasuring analyte using an isotachophoresis channel formed in a glasssubstrate, according to an embodiment;

FIG. 3 is a flow diagram that illustrates an example method forperforming isotachophoresis with gradients in an additive, according toan embodiment;

FIG. 4A through FIG. 4D are block diagrams that illustrate examplearrangements of an example isotachophoresis channel after steps of anexample method for performing isotachophoresis with gradients in anadditive, according to an embodiment;

FIG. 4E is a graph that illustrates example measurements correspondingto the arrangements of FIG. 4C and FIG. 4D, according to an embodiment;

FIG. 5A through FIG. 5D are block diagrams that illustrates examplepre-concentration of the analyte in a first zone, separation of similarspeed ions by size using a higher concentration of one additive in asecond zone, and detection using a lower concentration of anotheradditive in a third zone, according to an embodiment;

FIG. 6A through FIG. 6B are graphs that illustrate example dependence ofselectivity on polymer concentration as additive, according to anembodiment;

FIG. 6C is a graph that illustrates example effectiveness of denaturantas additive, according to an embodiment;

FIG. 7 is a graph that illustrates typical example isotachopherogram ofselective focusing of miRNA from total RNA, according to an embodiment;

FIG. 8A is a graph that illustrates an example calibration curve forabsolute quantification of miRNA using selective ITP, according to anembodiment;

FIG. 8B is a graph that illustrates an example quantitative assessmentof miRNA in Hepa1-6 and HeLa cell cultures before (low density) and atconfluence (high density), according to an embodiment;

FIG. 9A is a block diagram that illustrates a sequence specificmolecular beacon, used in some embodiments;

FIG. 9B and FIG. 9C are block diagrams that illustrate pre-concentrationof the analyte in a first zone, separation of similar speed ions by sizeusing a higher concentration of one additive in a second zone, anddetection using hybridization with molecular beacons based on higherconcentration of another additive in a third zone, according to anembodiment;

FIG. 10A is a graph that illustrates example demonstration of the ITPhybridization assay compared to a control, according to an embodiment;

FIG. 10B is a graph that illustrates the area of each peak of the tracesin FIG. 10A, according to an embodiment;

FIG. 11 is a graph that illustrates typical example fluorescenceenhancements f for ITP hybridization, according to an embodiment;

FIG. 12 is a graph that illustrates example results of the ITPhybridization assay for miRNA in liver tissue compared to a control,according to an embodiment;

FIG. 13A is a graph that illustrates example demonstration of ITPhybridization assay for detection and quantification of miR-122 inkidney and liver, according to an embodiment;

FIG. 13B is a graph that illustrates an example calibration curveresulting from interpolation of hybridization results from syntheticmiR-122 as a function of concentration, according to an embodiment;

FIG. 14A is a block diagram that illustrates pre-concentration of theanalyte and reporter ahead of a spacer ion in a first zone, andseparation of bound and unbound reporter ions by the spacer ions using ahigher concentration of one additive in a second zone, according to anembodiment;

FIG. 14B is a graph that illustrates example measurements of bound andunbound reporter ions in the arrangements of FIG. 14A, according to anembodiment;

FIG. 15A is a block diagram that illustrates pre-concentration of theanalyte and reporter, separation of bound and unbound reporter ionsusing a higher concentration of polymer sieve that fixes areporter-binding probe in a second zone, and a third zone of onlyanalyte-bound reporter ions in a third zone with no probe fixed in apolymer sieve, according to an embodiment; and

FIG. 15B is a graph that illustrates example measurements ofanalyte-bound control in the arrangements of FIG. 15A, according to anembodiment.

DETAILED DESCRIPTION

A method and apparatus are described for enhanced isotachophoresisassays using additives with spatial gradients. In the followingdescription, for the purposes of explanation, numerous specific detailsare set forth in order to provide a thorough understanding of thepresent invention. It will be apparent, however, to one skilled in theart that the present invention may be practiced without these specificdetails. In other instances, well-known structures and devices are shownin block diagram form in order to avoid unnecessarily obscuring thepresent invention.

Some embodiments of the invention are described below in the context ofseparating then detecting microRNA in a mixture with larger RNA strands,for example, as found in cellular material, using a microchannel.However, the invention is not limited to this context. In otherembodiments the same or different analyte is separated for purificationor detection or some other activity in microchannels or larger orsmaller channels. The proposed methods bring a substantial gain inflexibility or speed over traditional isotachophoresis approaches.

As used herein, an analyte is any collection of one or more kinds ofions that is targeted for separation from other species. Exampleanalytes include microRNA (miRNA), small interfering RNA, ribosomal RNA,messenger RNA, genomic DNA, proteins of interest, or small organic orinorganic molecules. Deoxyribonucleic acid (DNA) is a usuallydouble-stranded long molecule that encodes other shorter molecules, suchas proteins, used to build and control all living organisms. DNA iscomposed of repeating chemical units known as “nucleotides” or “bases.”There are four bases: adenine, thymine, cytosine, and guanine,represented by the letters A, T, C and G, respectively. Adenine on onestrand of DNA always binds to thymine on the other strand of DNA; andguanine on one strand always binds to cytosine on the other strand andsuch bonds are called base pairs. Any order of A, T, C and G is allowedon one strand, and that order determines the complementary order on theother strand. The actual order determines the function of that portionof the DNA molecule. Information on a portion of one strand of DNA canbe captured by ribonucleic acid (RNA) that also comprises a chain ofnucleotides in which uracil (U) replaces thymine (T). Determining theorder, or sequence, of bases on one strand of DNA or RNA is calledsequencing. A portion of length k bases of a strand is called a k-mer;and specific short k-mers are called oligonucleotides or oligomers or“oligos” for short. The term nucleic acid is used herein to refer to anyDNA molecule or RNA molecule or multi-nucleotide fragment or mixturethereof, whether a oligo or not.

A microfluidic channel, also called a micro channel herein, is a channelfor fluid flow with a width and height each less than 1000 microns (1micron=1 micrometer, μm, =10⁻⁶ meters). Microchannels can be etched inborosilicate glass using standard microfabrication processes.Microchannels can also be fabricated in fused silica, acrylic,polycarbonate, or polydimethylsiloxane.

As used herein, the leading electrolyte (LE) refers to the higheffective mobility co-ions rather than all constituents of a solution inthe channel before introduction of a sample and the trailingelectrolyte. Similarly, the trailing electrolyte (TE) refers to the loweffective mobility co-ions rather than the constituents of the sample orother constituents of the solution in the channel after the interfacewith the LE has passed.

An additive is a molecule different from both the leading electrolyte(LE) and the trailing electrolyte (TE); and the additive has a thirdeffective mobility that assures the analyte will encounter the additive.The additive can be positively charged, negatively charged, variablycharged or electrically neutral. The spatial gradient of the additiverefers to a non-zero change in concentration of the additive along thelength of the channel; and, the gradient can be positive, negative,constant or variable, e.g., ranging among positive, zero and negativevalues.

1. Overview

In this section, an overview of the methods and apparati are given. Moredetailed embodiments are described in later sections. One sectionpresents some detailed embodiments for quantification of microRNAcontent in a sample which also includes unwanted longer RNA strands, asfound for example in cellular material, or, more specifically, the totalRNA content of a cell. Another section presents embodiments in which themicroRNA is extracted using additive gradients and then hybridized withmolecular beacons that fluoresce when particular sequences of nucleotidebases are encountered. In other embodiments, different methods are usedto introduce gradients in one or more additives, or reporters such asmolecular beacons bound to the analyte are separated from such reportersthat are not bound to the analyte by varying the properties of a polymersieve or by introducing spacer ions or both. Below, references arecited, each of which is hereby incorporated by reference as if fully setforth herein, except so far as the terminology is inconsistent with theterminology used herein.

The additive can be used to perform any function duringisotachophoresis, including preferentially entangling long molecules,preventing or encouraging folding (e.g., secondary and tertiarystructures) of molecules, or labeling molecules with one or morefluorophores, among many others. Because of the spatial gradient, thefunction of the additive is performed to greater or lesser degree atdifferent locations along the channel, depending on the concentration ofthe additive at that location.

By virtue of the effective mobility of the additive by definition, theanalyte sequentially moves through the different concentrations of theadditive as the analyte moves along the channel during isotachophoresis.Thus, the analyte experiences the functions of the additivecorresponding to the additive's concentration in sequence as the analytemoves through the channel. The ITP channel becomes like an assembly lineto process a sample with a sequence of operations.

FIG. 1A is a diagram that illustrates an example isotachophoresischannel 110 with a spatial gradient of an additive before application ofan external electric field, according to an embodiment. The channel 110connects an input port 112 to an output port 114 and is disposed betweentwo electrodes 122 and 124. Before an external electric field is appliedbetween the electrodes, the channel and output port 114 are filled withthe LE mixed with one or more additives.

According to various embodiments, the concentration of one or moreadditives changes along the length of the channel, either continuouslyor abruptly, thus setting up a spatial gradient of additiveconcentration. Any method may be used to establish the spatial gradient,such as sequentially introducing different mixtures of LE and additivethrough the input port 112, or introducing different concentrations ofadditive or additive and LE mix through side ports (not shown)distributed along the channel 110, or by differentially polymerizingmonomers or short polymers, among other approaches. The spatiallynon-uniform concentrations of additives are represented in FIG. 1A byzone 1 though zone n with corresponding additive concentrations C1through Cn, called the additive concentration gradient 130, hereinafter.In various embodiments, the concentrations of various additives increaseor decrease with distance or both, so, in some embodiments, theconcentration gradient is neither constant nor monotonic. In someembodiments, each zone is relatively homogenous with discreteconcentrations of one or more additives and sharp additive concentrationchanges at zone boundaries. In some embodiments, the spatial gradient inthe channel is smooth and slowly changing and each zone has a range ofconcentrations of one or more additives without sharp additiveconcentration discontinuities at the zone boundaries.

Any additive that provides a useful function during ITP may be used invarious embodiments. An example additive is a polymer sieving matrix(e.g. for the sieving of nucleic acids) such as polyvinylpyrrolidone,polyethylene glycol, polyethylene glycol, hydroxyethyl cellulose,poly(N,N-dimethylacrylamide), pluronic F127, agarose, linearpolyacrylamide, acrylamide etc. Another example additive is a denaturingagent (e.g. for preventing secondary structures of nucleic acids, suchas folding and hybridizing to a second strand, which interferes withsieving) such as formamide, urea, N,N-dimethylformamide, acetic acid,guanidinium chloride etc. a surfactant such as Triton X-100, TritonX-200, Tween 20, Tween 80, saponin (e.g. for cell lysis). Anotherexample additive is a fluorophore, such as rhodamine B, rhodamine 123,rhodamine 6G, fluorescein dyes, Alexa Fluor dyes, Cy dyes, Bodipy dyes,DyLight dyes, green fluorescent protein, red fluorescent protein.Another example additive is a labeled probe such as molecular beacons,Taqman, oligonucleotide probe end-labeled with a fluorophore and, insome embodiments, a fluorescence quencher at the other end. Anotherexample additive is acrylamide-modified oligo (e.g., 5′ modificationAcrydite). As used herein, a reporter is any molecule that binds to ananalyte to make the analyte more detectable. Reporters include afluorophore, a quantum dot, and labeled probes, among others. Anotherexample additive is a reagent for interacting with the segregated orbound analyte.

FIG. 1B is a diagram that illustrates an example isotachophoresischannel with a spatial gradient of an additive after application of anexternal electric field, according to an embodiment. Upon application ofthe external electric field 126 (shown in the direction of the movingions), the target analytes of the sample 160 focus at the LE-TEinterface and propagate through the sequence of relatively stationaryzones, from left to right. In some following illustrations, the electricfield direction is depicted as directed from positive charge to negativecharge and is therefore directed opposite to the direction of flow ofthe ions, e.g., for negatively charged ions (anions).

FIG. 1C is a graph 180 that illustrates an example electric field 190 inthe channel at a time that corresponds to FIG. 1B. The horizontal axis182 is distance along the channel (relative units); and the verticalaxis 184 is electric field in the channel (relative units). The mobilityof the TE is less than the mobility of the LE, so the conductivity andthe electric field strength in the TE are, respectively, lower andhigher than those of the LE. A steep electric field gradient (dashedline) is created at the ITP interface traveling at velocity U_(ITP)between TE (dotted line) and LE (solid line); other electric fieldgradients in the channel are created by the difference in conductivitiesof additives in discrete zones. A target analyte ion in the sample whichencounters the higher electric field of the TE, speeds up and focuses atthe interface. Similarly, a target analyte ion in the sample whichencounters the lower electric field of the LE, slows down and returns tothe focus at the interface as this interface propagates from left toright.

As the analyte encounters each additive concentration zone, the analyteinteracts with the additives in that zone according to the effect of theconcentration of the additive in the encountered zone. Thus, eachadditive operates most effectively on a component of the sample along aportion of the channel where the concentration of the additive is in aparticular range of concentrations.

In some embodiments, the channel is a microchannel.

2 Apparatus

FIG. 2A is a block diagram that illustrates an example isotachophoresischannel 210 formed in a glass substrate, according to an embodiment. Atleast some experiments described below were performed in anoff-the-shelf borosilicate glass microfluidic chip (model NS260, CaliperLS, Mountain View, Calif.) depicted in FIG. 2. A scale bar 201 indicatesa length scale of 1 centimeter (cm, 1 cm=10⁻² meters). The channel 210connects an input port 212 to output (exit) port 214. Along the channel,six reservoir ports 222, 223, 224, 225, 226, 227 are connected to thechannel at corresponding T-junctions. Each reservoir is cylindrical 1.8mm in diameter and 1 mm in depth. The reservoir ports (collectivelyreferenced hereinafter as reservoir ports 220) are each connected tochannel 210 by 12 micron deep, 44 micron wide wet-etched borosilicateglass microchannels. These reservoir ports allow for forming multiplezones of additive concentrations. For example, three additiveconcentration zones are provided in some embodiments described below bycontacting a first additive mixture with the LE (designated mixture LE1)at ports 212, 222, 223 up to point A, and contacting a second additivemixture with the LE (designated mixture LE2) at ports 224, 225, 226 upto point B, and contacting a third additive mixture with the LE(designated mixture LE3) at ports 222, 214 up to the output port. Meansfor establishing particular concentration gradients of particularadditives are described in more detail below with reference to variousembodiments.

FIG. 2B is a block diagram that illustrates an example apparatus 230 formeasuring analyte using an isotachophoresis channel formed in a glasssubstrate, according to an embodiment. Besides the glass chip 200depicted in FIG. 2A, the apparatus 230 includes a high voltage powersupply 232 having an electrical ground (GND) terminal 234 and aselectable voltage at a high voltage (HV) terminal 236. The apparatusfurther includes microscope 240 with an objective lens 242 and opticalfilter cube 244, a diode laser 248, optical detection including apinhole 252, lens 254 and photomultiplier tube (PMT) 256. PMT data weredigitized and processed in a computer, such as PC-DAQ 260, describedbelow.

For example, in some experimental embodiments, fluorescence data wasacquired using an inverted epifluorescence microscope 240 model EclipseTE200 from Nikon of Japan, equipped with a laser diode illumination 248(642 nanometer, nm, wavelength, 1 nm=10⁻⁹ meters) model Stradus 642 fromVortran of Sacramento, Calif. Light was filtered using a standard Cy5cube 244 (exciter/emitter 630/695 nm) model XF110-2 from Omega Opticalof Brattleboro, Vt., and focused though a 60× water immersion objective242 (N.A.=1.0) model Fluor from Nikon of Japan. To reduce noise from outof focus light sources in the returned fluorescent light, a customconfocal assembly was built by placing a 150 micron diameter pinholeprovided as a mounted precision pinhole from Edmund Optics ofBarrington, N.J. at the image focal plane. Fluorescence intensity wasmeasured using a lens 254 and PMT 256 model H7422-40 from HamamatsuPhotonics of Japan, with voltage set to 900 volts (V). The PMT signalwas converted using an amplifier/converter unit model C7319 fromHamamatsu of Japan, and was filtered with a simple low pass RC circuit(RC=1.2 ms). The resulting voltage signal was acquired at personalcomputer 260 with a DAQ card model NI USB-6211 from National Instrumentsof Austin, Tex. controlled with Matlab software from The Mathworks ofNatick, Mass. In some experimental embodiments, measurements werecollected at 250 kilo-samples per second (kS/s, 1 kS/s=10³ samples persecond) data rate; and, a 4,000 points moving average was applied to thesignal for analysis. In some embodiments measurements are performed at90 kS/s and filtered with a 1500 point moving average. In someembodiments, to characterize a fluorescence peak, the voltage trace wasanalyzed by fitting a Gaussian function to the ITP peak. Fluorescenceintensity was then calculated by integrating the raw data under the fitover three standard deviations.

To produce the temporally changing spatial profiles (spatiotemporalplots) in some embodiments, a mercury lamp is used for illumination inlieu of diode laser 248, with blue/green filter (Omega XF115-2) asfilter 244, 4× objective lens as objective 242, and PrincetonInstruments Coolsnap charge coupled device (CCD) camera to image alength of the channel in lieu of pMT 256. Winview32 software was used tocontrol the camera from a PC. In some embodiments, a 488 nm collimateddiode light source from Thorlabs of Newton, N.J. was used forillumination, in lieu of diode laser 248. A 4× objective lens (N.A.=0.2)model Plan Apo from Nikon of Japan was used as objective 242. Imagesalong a length of the channel were acquired using a cooled CCD cameramodel Cascade 512F from Photometrics of Tucson, Ariz. controlled withWinview32 software from Princeton Instruments of Trenton, N.J. operatingon PC 260 in lieu of PMT 256.

3 Method

FIG. 3 is a flow diagram that illustrates an example method 300 forperforming isotachophoresis with gradients in an additive, according toan embodiment. Although steps are depicted in FIG. 3 as integral stepsin a particular order for purposes of illustration, in otherembodiments, one or more steps, or portions thereof, are performed in adifferent order, or overlapping in time, in series or in parallel, orare omitted, or one or more additional steps are added, or the method ischanged in some combination of ways.

In step 301, the analyte to be measured is determined. For example, insome embodiments, the analyte is a microRNA (miRNA) molecule, with onthe order of ten nucleotides, or a miRNA molecule including a particularsequence of nucleotides, as described for various embodiments in moredetail below.

In step 303 the TE, LE, spacer ions, and one or more additive gradientsare selected based on the relative motilities of the analyte and anycontaminants to be separated from the analyte. For example, in theillustrated embodiments, analyte miRNA is separated from long RNAcontaminants, or is separated from miRNA with incorrect sequences thatare regarded as contaminants. In some embodiments, spacer ions are used,as described in more detail below. In some embodiments, spacer ions areused, as described in more detail below. Spacer ions are described inUnited States Published Patent Application US-2010-0084271-A1, publishedon Apr. 8, 2010, the entire contents of which are hereby incorporated byreference as if fully set forth herein, except as the terminology usedtherein is not consistent with the terminology used herein. Spacer ionsare selected to have a mobility that is between the mobilities of one ormore molecules that react (reactants) and the mobility of a productmolecule of those reactants. For example, spacer ions are used toseparate a miRNA molecule and a probe specific to a particular sequenceof nucleotides, from the hybrid of the probe with an miRNA molecule thatmatches the particular sequence, as described in more detail below.

Additives are selected to prepare the analyte for measurement, e.g.,reporters, including sequence specific probes, denaturants to reducesecondary structure, enzymes to foster interaction, accelerators thatincrease rate of hybridization, polymers or gels to slow the movement oflonger molecules, reactants to form or condition the analyte, amongothers. Additive mobility is selected to ensure that the analyte will beexposed to the additive gradient during the transit of the analyte frominput port to output port.

In step 305, the channel is prepared for initial concentrations of theLE and one or more additive gradients. For example, various mixtures arecontacted to the reservoir ports 220 of glass chip 200. In someembodiments, step 305 includes differentially polymerizing a monomer orsmall polymers, or both, along the channel, as described in more detailin a later section. The differential polymerization causes differencesin cross-linking and forms a gel fixed to one or more channel wallsdifferentially along the length of the channel. In some embodiments withpolymerized gels, a surface modification is applied to the microchannelprior during step 305 before introducing the one or more mixtures. Forexample, in some embodiments, the channel is coated with3-(trimethoxysilyl)propyl methacrylate. With this coating in place,after polymerization, the gel becomes covalently bound to the channelsurface, which prevents the gel from slipping in the channel (e.g. dueto electro-osmotic flow, or shrinking).

In step 309, a sample containing analyte or analyte precursor iscollected and prepared and contacted to the TE and any spacer ions toform a sample mixture. In step 311, the sample mixture is contacted tothe input port of the channel, e.g., 112 or 212. In some embodiments,steps 309 and 311 are performed together, and the sample mixture isformed at the input port 112 or 212.

In step 313 the channel is subjected to an electric field to motivateions in the channel. For example, external electrodes are charged insome embodiments. In some embodiments, an electrode inside the inputport is electrically grounded and an electrode in the output port ischarged to a high voltage.

In step 315, an indicator of the analyte is measured at a viewing pointnear the output (exit) port of the channel during a time window thatcorresponds to the passage of the analyte. In some embodiments, themeasurements are made for a longer duration to measure conditions beforeand after the passage of the analyte. In some embodiments, the viewingport is moved along the channel, either by motion of the microscope 240or by motion of the glass chip 200 or some combination. The indicator isany measurable that is related to the concentration or state of theanalyte, such as fluorescent emissions from a reporter or from theanalyte itself. In various embodiments the indicator is opticaltransmission, absorption, spectra thereof, magnetic signal, voltage,among others.

In step 317, the presence/concentration of the analyte is determinedbased on the measurements. For example, an area under a measuredfluorescence peak is converted to analyte concentration based oncalibration curves formed by performing steps 305 to 315 with sampleshaving known concentrations of analyte).

In step 319, the condition of a subject from which the sample was takenis determined based on the presence/concentration of the analyte. Forexample, the subject is determined to have abnormal liver function basedon miRNA sequences that are not normal for liver cells. For example, asample of nucleic acids extracted from blood or urine of the subject isdetermined to have show presence/absence of a pathogen (bacterial orviral) or toxin, e.g., diagnosing urinary tract infections based ondetecting e. coli rRNA using molecular beacon.

Method 300 provides an assay for the detection and quantification ofanalytes, such as miRNA targets in total RNA samples. The assay is basedon an ITP process which selectively focuses and accentuates detection ofanalyte, e.g., via hybridizations with molecular beacons (MB). In someembodiments, described in more detail in the next section, ITPhybridization is a fast (<2 minute), low component cost (about $50 perchip, standard epifluorescence microscope and power supply, about $0.50of reagents per 100 runs), and sensitive (down to 3,000 copies per cell)microfluidic method for miRNA profiling that requires small amounts ofsample (100 nanograms, ng, 1 ng=10⁻⁹ grams, of total RNA) with aboutthree decade dynamic range. Its speed, automation and low sampleconsumption make it an attractive alternative to PCR or northern blotanalysis. It is anticipated that further optimization of ITP and MBchemistries and dynamics would significantly enhance sensitivity andreach the 100 copies per cell level. It is also anticipated that ITPhybridization can be extended to the detection and quantification of anytype of nucleic acids, for example messenger, ribosomal RNA or genomicDNA. This has been shown in a recent publication, see Bercovici et al.,Analytical Chemistry 2011 for work on diagnosing urinary tractinfections based on detecting e. coli rRNA using molecular beacon

4. Example Embodiments 4.1 Polymer Gel Additive

In some embodiments, discrete gel sieving matrix concentration gradientsare used to separate analytes, thus coupling isotachophoresis (ITP) withspatially varying capillary gel electrophoresis (CGE). Ultraviolet (UV)light is used to create a discrete gel region within a microchannel,thus partitioning the channel longitudinally into two zones. In someembodiments, long polymers in solution serve as the sieving matrix. Inthe embodiments described here, however, a gel polymerized in place canform more and stronger cross-links and become fixed to one or more wallsof the channel to form a sieving matrix that is fixed and abruptlychanging with distance along the channel. By using photolithographytechniques, the gel can be formed along any arbitrary portion of amicrochannel.

In a first zone, analytes in free solution focus in peak mode ITP,resulting in a single sharp peak. Upon entering the gel (sieving matrix)region, analyte mobility decreases below TE mobility. Analytes thereforeseparate according to their effective mobilities within this region.Separation relies on significant retardation of large molecules (e.g.DNA) as compared to small ions (e.g. TE and LE ions). These interactionsare dependent on several factors, including analyte structure and size.This technique is demonstrated by separating a double stranded DNAladder containing oligonucleotides ranging from 100 nucleotides (nt) to12,000 nt in a 6% polyacrylamide gel matrix.

FIG. 4A through FIG. 4D are block diagrams that illustrate examplearrangements of an example isotachophoresis channel after steps of anexample method for performing isotachophoresis with gradients in a geladditive, according to an embodiment. FIG. 4A and FIG. 4B showarrangement of channel 110 during portions of channel preparation step305 of method 300. In FIG. 4A, the channel 110 is filled from the outputport 114 with a mixture 402 comprising a buffer that contains theleading electrolyte (LE), acrylamide/bisacrylamide monomer and across-linker, and a photo-initiator. In an experimental embodiment, thefollowing was used:—50 mM Hydrochloric Acid (HCl) as LE,—100 mM Tris asa counterion,—6% (w/v) acrylamide as monomer—3.3% (w/w) bisacrylamide ascrosslinker—0.2% (w/v) VA-086 as a photoinitiator. The channel 110 isfilled with the help of a vacuum 404 contacted to input port 112.

In FIG. 4B, a photo-mask 408 is used to cover portions of the channel110, allowing spatially-selective exposure to polymerizing light, e.g.,UV light 410, thus photo-patterning a desired portion with fine spatialresolution and an abrupt boundary. As a result of the polymerization, apolymer gel 412 is fixed in the channel 110. Although this examplepolymerizes a monomer with UV light, in other embodiments the gel ispolymerized in place using other cross-linkers, polymerizationinitiators and polymerizing energy (e.g., heat, visible light, amongothers). In an experimental embodiment, the channel was illuminated fora period of 6 minutes and a distance of 1.5 cm using a mercury lamp witha 50:50 beam-splitter. The intensity of the UV light is 300 mW/cm² overa uniform beam area of 0.28 cm².

In step 311, a buffer containing the trailing electrolyte (TE) and theconditioned (e.g., fluorescently-labeled) analytes or intercalating dyeof choice are added to one of the wells, e.g., to input port 112,replacing and contacting the LE buffer. In step 313, an electric fieldis applied using electrodes 236, 234 in the wells. ITP is thus carriedout. As shown in FIG. 4C, ITP focuses the analytes in a migratinginterface 430 between the fast LE ions in mixture 402 and the slow TEions in buffer 422. The formation of a single focused peak is observedas the analytes approach the region with the polymer gel 412. FIG. 4Ddepicts the separation of the DNA molecules by size in the polymer gel.Upon entrance in the region of the polymer gel 412, the rapid resolvingof the focused peak into multiple, lower intensity bands 432 isobserved, indicating successful separation of different sizeoligonucleotides through CGE. This separation is a result of theanalytes' interactions with the gel, which has a more significantretardation effect on larger DNA. This allows the TE, an ion, which isnot retarded by the gel to the same extent, to progressively pass thesebands, thus separating them. Upon exiting the gel region (not shown),the bands re-focus into one peak at the LE-TE interface, allowing forin-line (downstream) processes to be implemented.

FIG. 4E is a graph 450 that illustrates example measurementscorresponding to the arrangements of FIG. 4C and FIG. 4D, according toan embodiment. The horizontal axis is distance along the channel 110 inmillimeter (mm, 1 mm=10⁻³ meters); and, the vertical axis is time inseconds. At each time (e.g., about every 0.2 seconds) an image of thefluorescence in a 2 mm portion of the channel is collected. The polymergel occupies a portion of the channel from about 0.9 mm to about 1.3 mm.The single bright fluorescent peak 460 observed in the first 0.5 mm andfirst second of the ITP clearly gives way to multiple fainter peaks 462in the region of the gel at times after about 2 seconds.

4.2 Quantification of microRNA

MicroRNAs (miRNAs) are a growing class of small, noncoding RNAs (17-27nucleotides) that regulate gene expression by targeting mRNAs fortranslational repression, degradation, or both. These molecules areemerging as important modulators in cellular pathways such as growth andproliferation, apoptosis, and developmental timing. To date, thousandsof miRNAs have been identified in organisms from viruses to primatesthrough cloning and sequencing, or computational prediction based onstrong conservation of miRNA sequence motifs.

The most popular and well-established miRNA profiling methods areadapted from traditional nucleic acid analysis techniques. These includenorthern blot, microarrays, sequencing and reverse-transcription PCR(RT-PCR). Microarrays and sequencing platforms have high throughput butrequire significant instrumentation, amount of sample (about 5 μg oftotal RNA), are time consuming and require pre-amplification whichyields significant sequence bias. RT-PCR has high dynamic range and issensitive but has low throughput and is less specific than standard PCR.Lastly, northern blot has moderate sensitivity and allows for lengthdiscrimination of sequences, but remains time consuming and requireslarge amounts of sample (often>1 μg of total RNA). Northern blottingconsists of gel electrophoresis for separation of total RNA withsubsequent transfer to a nitrocellulose membrane, followed byhybridization with a radioactively labeled probe visualized with ascintillation counter.

The technique of spatial gradient additives has been used for thesensitive and selective absolute quantification and isolation of miRNAin an RNA sample, as described in greater detail below. In this example,a three-zone ITP channel with varying concentrations of largelynon-ionic polymer sieving matrix and denaturant and label. These threeserial zones allowed for consecutive pre-concentration, selection andquantification of miRNA.

FIG. 5A through FIG. 5D are block diagrams that illustrates examplepre-concentration of the analyte in a first zone, separation of similarspeed ions by size using a higher concentration of one additive in asecond zone, and detection using a lower concentration of anotheradditive in a third zone, according to an embodiment. FIG. 5A shows apreconcentration first zone 502, an miRNA selection second zone 504 anda detection third zone 506, as well as the direction of externalelectric field 126. Concentration of polymer 508 in solution isindicated by cross hatching in which wider separation indicates lowerconcentration of the polymer in solution. The polymer concentrationincreases to a maximum in the second zone 504. In the preconcentrationzone 502 the first LE mixture (e.g., LE1 described above with referenceto FIG. 2A) allows both miRNA and RNA ions to focus at the interface 512between TE and LE ions. In the miRNA selection zone 504 the second LEmixture (e.g., LE2 described above with reference to FIG. 2A) allowsmiRNA in a leading focused interface 516 and causes RNA ions to de-focusin a following population peak 514, with denaturing agents enhancing theseparation. In the detection zone 506 the third LE mixture (e.g., LE3described above with reference to FIG. 2A) labels the miRNA in theleading focused interface 516 without hindrance by denaturing agents.

For example, in this embodiment to quantify miRNA, one additive is apolymer in solution. The additive is used as a sieving matrix thatserves to sieve more effectively as the concentration of the polymerincreases. As the small target analyte (e.g, miRNA) moves from a regionof low sieving matrix concentration to one of high sieving matrixconcentration, it stays focused. However, molecules (such as RNA) oflonger-than-targeted length have a mobility which drops below that ofthe TE in the presence of high concentrations of the sieving matrix, andso get left behind and are no longer focused in the zones after passingsuch a high concentration zone.

The sieving matrix concentration is not uniform throughout the channelbecause it is desirable to have low sieving matrix concentration in thefirst zone to allow a less specific, preliminary sorting of RNA fromlonger RNA while still maintaining a relatively high value of RNAmobility. The latter maintains a high rate of accumulation of RNA intothe ITP focus zone (the interface between TE and LE). This allows forhigher accumulation rate in a shorter channel length. However, once thispreliminary sorting and high rate accumulation are accomplished, thesample propagates into a second zone. The second zone uses highersieving matrix concentration to achieve separation of miRNA fromslightly longer RNA which has slightly lower mobility in local sievingmatrix concentration.

To allow the sieving matrix to more effectively interact with and slowdown the longer RNA chains, a denaturing agent is also included in thefirst and second zones. The denaturing agent mitigates tight folding ofthe RNA chains so that more interaction occurs with the sieving matrixand sieving can be more effective. By the third zone, the longer RNAchains are no longer traveling in the focused sample at the interface;and, denaturing conditions are much less important. Furthermore, a labelis included as an additive in the third zone, and high concentration ofthis denaturing agent interferes with the binding of this label to themiRNA. Thus label concentrations are increased in the third zone anddenaturing concentrations are decreased. The concentration of denaturantis decreased in the third zone because the fine separation of short RNAfrom slightly longer RNA is not required in the third zone (since theslightly longer RNA have been left behind a significant distance). Thishelps improve the focusing dynamics of the focused RNA to improvesensitivity of the assay.

Therefore, in this example embodiment, the sieving additive is low inthe first zone, and high in the second zone, and again lower in thethird zone. The denaturing agent additive concentration is high in thefirst two zones and low in the third zone. And the label additiveconcentration is zero in the first zone and significant in the secondand third zones.

FIG. 5B is a block diagram that depicts TE ions 522, LE ions 524, miRNAions 526 and large RNA ions (with greater than 40 nt) 528, each withcorresponding mobility magnitudes represented by the symbol“V,” in eachof the three zones 502, 504 and 506. In zone 502 both miRNA 526 and RNA528 have mobilities between the mobility of TE ions 522 and the mobilityof LE ions 524. In zone 504, the mobility of RNA falls below that of theTE ions 522 and those molecules fall out of focus at the interface. Inzone 506 the remaining miRNA ions 526 take up a fluorescent label (notshown) and stay focused between the TE ions 522 and the LE ions 524.

Thus, in some embodiments, a first additive comprises a polymer thatprovides a sieving matrix. Further, in some embodiments, the analyte isa nucleotide and a second additive comprises a denaturing agent.Furthermore, in some embodiments, a third additive comprises afluorescent label.

This is accomplished in an experimental embodiment with the followingexperimental procedures. During step 305 the channel is prepared in theoff-the-shelf chip design (model NS260, Caliper LS, Mountain View,Calif.) as shown on FIG. 2A. Before each set of experiments, the chip isfirst preconditioned by rinsing the channels successively with 100milliMoles (mM, 1 mM=10⁻³ Moles) sodium hydroxide for 5 minutes (min),deionized (DI) water for 1 min, 100 mM hydrochloric acid for 5 min, andDI water for 1 min. The different LE mixtures are then added toreservoirs 212, 220 and 214 as follows, 5 microliters (ml, 1 ml=10⁻⁶liters) of LE1 into each of ports 212, 222, 223; of LE2 into each ofports 224, 225, 226; and of LE3 into each of ports 227 and 214. A vacuumis applied to reservoirs 223 and 227 for 5 min. These initial rinsingand filling steps are useful to reduce and stabilize electroosmotic flowin the borosilicate chip.

The LE mixtures all contain Tris hydrochloride (pH=8.0), urea,Polyvinylpyrrolidone (PVP). For RNA quantitation, we used SYTO RNAselectdye, except for the spatial temporal diagrams of FIG. 5C and FIG. 5D,described below, where we used SYBR Green II. The TE is a solution of92.5% v/v formamide containing Tris and Caproic acid. We use urea in theLE1 and LE2 zones to increase separation resolution for greateraccuracy, but this significantly decreases fluorescence of the RNAstain. Consequently, we use reduced denaturing conditions (low C_(d)) inLE3, so this last section acts as a detection zone.

Before each experiment, all reservoirs are rinsed with DI water. Thedifferent LE mixtures are added to reservoirs 212, 220 and 214 asdescribed above and again a vacuum is applied to reservoir ports 223 and227 for 2 min. Applying vacuum at port 223 creates an interface betweenLE1 and LE2 at the intersection A and applying vacuum at port 227creates an interface between LE2 and LE3 at the intersection B. Thevacuum is then released, the input port 214 is rinsed and emptied, andcontacted with the mixture of TE and sample. Finally, a 3 kV potentialdifference is applied between output port 214 and input port 212 tostart the ITP process and a stop voltage is applied after the ITPinterface has reached the detector using the high voltage power supply232.

In an experimental demonstration of this process, a 22 nt analyte and 60nt long RNA contaminant in a sample were focused in zone LE1 and the 60nt long RNA contaminant was selectively defocused in zone 2. Thesequences of the nucleic acids used in these experiments are given inTable 1.

TABLE 1 Sample nucleic acids in various experiments. SEQ. IDLength (name) NO. Sequence (5′ to 3′) 23 nt  1 CAAAGUGCUUACAGUGCAGGUAG(miR-17) 40 nt 2 CUGUGACACUUCAAACUCGUACCG UGAGUAAUAAUGCGCC 60 nt 3CAUUAUUACUUUUGGUACGCGCUGU GACACUUCAAACUCGUACCGUGAGU AAUAAUGCGC 22 nt  4UCGUACCGUGAGUAAUAAUGCG (miR-126) 22 nt  5 CGCAUUAUUACUCACGGUACGA(complimentary to miR-126)

FIG. 5C is a spatiotemporal plot 530 of the population peaks of the twoRNA species in the transition from zone LE1 to LE2; and FIG. 5D is aspatiotemporal plot 550 of the population peak of the smaller 22 nt RNAspecies in the transition from zone LE2 to LE32. Plot 530 has horizontalaxis 532 representing distance along channel and a vertical axis 534representing time. Plot 550 has horizontal axis 552 representingdistance farther along the channel and a vertical axis (not shown)representing a later time. Both plots share distance and time scale bars536 representing 1 second vertically and 0.5 mm horizontally.Illumination is indicated by grayscale given by scale bar 538. Thefluorescent signal at the detection point is represented by trace 580 oninsert graph with horizontal axis 572 representing signal strength andvertical axis 584 representing cross channel distance. The amount ofanalyte is represented by the area under a gaussian fit to the signalthat is three standard deviations (3σ) wide centered on the peak.

FIG. 5C shows channel-width-averaged fluorescence intensity (invertedgrey scale) versus axial channel distance and time. Here, a mixture of22 nt and 60 nt long RNA focus in LE1, but only the 22 nt RNA remainsfocused in LE2. FIG. 5D shows transition of a 22 nt RNA focused peakfrom low fluorescence in LE2 to significantly larger fluorescence inLE3. Recall that the LE3 zone has reduced denaturant concentrationenabling higher fluorescence for sensitive quantification of theselectively focused miRNA.

To perform exquisitely selective miRNA focusing, we first chose a TEwhose mobility was smaller than the mobility of short nucleic acids (forzero C_(p)), and chose nominal values for C_(p) in LE1 and 2. We thenperformed a series of experiments with increasing C_(p) (and decreasinglocal RNA mobility) in LE2. Such titration allowed tuning of the cut-offfocusing length (length below which RNA focuses).

In FIG. 6A, results are shown of three sets of titration experimentsusing 23 and 40 nt synthetic oligoribonucleotides and yeast tRNA. A 40nt long synthetic oligo was used to simulate RNA longer than miRNA, andtRNA (80 nt in average) to verify that highly abundant short RNAs withstrong secondary structures do not interfere with the measurement. Thetitration aimed at finding the C_(p) to focus miRNA but reject 40 merand tRNA. FIG. 6A and FIG. 6B are graphs 600 and 620, respectively, thatillustrate example dependence of selectivity on polymer concentration asadditive, according to an embodiment. In FIG. 6A, the horizontal axis602 is concentration of polymer PVP in solution in the LE2 mixture; and,the vertical axis 604 is fluorescence intensity in arbitrary units. Inthese experiments 23 nt and 40 nt long RNA and tRNA as a sample withanalyte and contaminant, respectively, were dissolved in the TE. Totalintensity in the focused zone is reported for increasing polymerconcentration in LE2. All RNA focus at 3% weight/volume (w/v) PVP. At 5%w/v and above, significant focusing of the 23 nt RNA is still observedwhile both 40 nt and tRNA are rejected.

In FIG. 6B, the horizontal axis 622 is ratio of concentration of the 40nt oligo to the 23 nt oligo; and the vertical axis indicates two valuesfor the polymer PVP concentration in solution. At 4% w/v PVP, F₄₀/F₂₃ isgreater than 0.6, but drops down to 0.12 at 5.5% w/v. This led to thechoice to use 5.5% w/v PVP in LE2 for selective focusing of miRNA.

For all three RNAs, the amount of focused RNA gradually decreased withincreasing PVP initial concentration in LE2. This is consistent with aglobal decrease of nucleic acid electrophoretic mobility, and associateddecreased flux of RNA to the ITP interface. At 3% w/v PVP, there wassignificant focusing of all three RNA types. Increasing PVPconcentration to 4% w/v resulted in defocusing of tRNA, shown by thedrop in tRNA signal to the baseline value. 40 nt RNA was rejected at 5%w/v PVP. Meanwhile, the amount of 23 nt long RNA remained significant atall concentrations. In particular, at 5.5% w/v PVP, the measured(baseline) fluorescence intensity of the 40 nt RNA case was only 12% ofthe fluorescence of miRNA, but exceeded 60% of miRNA at 4% w/v. Weattribute most of the residual fluorescence at 5.5% w/v to contaminationand synthesis byproducts remaining after purification of the 40 ntoligo. This titration shows refined selective focusing of miRNA with anLE2 with 5.5% w/v PVP and an RNA cutoff length between about 24 and 39nt.

FIG. 6C is a graph that illustrates example effectiveness of denaturantas additive, according to an embodiment. The horizontal axis indicatesniR-126 and hybridized with a complementary sequence (duples). Thevertical axis is fluorescence intensity in arbitrary units. This showsresults of selective focusing of miR-126 (5 pg·μl⁻¹ in TE) and of anequimolar mixture of mir-126 and its complementary RNA (each at 2.5pg·μl⁻¹). There is no significant difference between fluorescence ofmiR-126 and of the duplex, showing no bias due to base pairing.Uncertainty bars 646 a, 646 b represent 95% confidence interval. Theresults show that secondary structure of tRNA had no discernable effecton assay selectivity, e.g., that the amount of denaturant is sufficientto prevent secondary structure.

FIG. 7 is a graph 700 that illustrates typical example isotachopherogramof selective focusing of miRNA from total RNA, according to anembodiment. The horizontal axis 702 is time in seconds a the viewingport near the output (exit) port. The vertical axis 704 is fluorescencein arbitrary units. The trace 710 is a measurement at the viewing point.The first arrival is the focused miRNA at peak 712. A much later arrivalis a much less focused peak of longer RNA molecules, such as tRNA peak714. The sharp peak 712 at t=92 s corresponds to the ITP focused miRNA.This peak is approximately Gaussian with characteristic width around 20ms. The peak(s) 714 at larger migration times (t>100 s) corresponds tolonger RNA molecules, most likely transfer RNA (tRNA). For each run, aGaussian fit on the miRNA peak was performed and the signal wasintegrated over three standard deviations, centered on the peakposition. This run corresponds to a typical miRNA quantitation run forHepa1-6 cells (5 ng·μl−1 in the TE). Before continuing, we note weperformed similar measurements on degraded RNA samples, and these showedsignificant tailing of the miRNA peak, a dispersed tRNA peak, andoverlap between these. Degraded RNA samples also showed abnormally highlevels of focused short RNA compared to higher quality preparations.Under such conditions, degraded RNA likely produced fragments shorterthan the ITP cutoff length, resulting in highly upward biasedquantitation of miRNA. To avoid this bias, we systematically obtainedRNA integrity numbers (RIN, measured at the Stanford PAN facility) forall samples and performed measurements exclusively on samples with RINgreater than 9.0, which exceeds recommendations for miRNA analysis.

Calibration curves were determined by diluting the miRNA reference inthe TE at relevant concentrations. FIG. 8A is a graph that illustratesan example calibration curve for absolute quantification of miRNA usingselective ITP, according to an embodiment. The horizontal axis 802 ismiRNA concentration in picograms per microliter (pg/μL, 1 pg=10⁻¹² gramsand 1 mL=10⁻⁶ liters). Points 812 are measurements and trace 814 is alinear fit. The miRNA concentration varied between 0.3 and 30 pg·μL⁻¹.Negative controls (experiments with no miRNA in the TE) yielded areproducible fluorescent signal at the 0.1 pg·μL⁻¹ level. This residualfluorescence was likely due to contamination of stock chemicals asobserved routinely by us and others using ITP.

To demonstrate the efficacy and utility of this ITP assay in a stronglyrelevant biological application, we quantified global miRNA levels insubconfluent and confluent cell cultures. Hwang, H. W.; Wentzel, E. A.;Mendell, J. T. Proceedings of the National Academy of Sciences of theUnited States of America 2009, 106, 7016-7021 (hereinafter, Hwang etal.) showed that cell-cell contact activates miRNA biogenesis, resultingin greater miRNA abundance in densely-grown cultures of various celllines. Herein is provided further independent evidence for this effectusing the ITP-based selective quantitation by measuring miRNA abundancein HeLa and Hepa1-6 cell cultures before and at confluence. FIG. 8B is agraph that illustrates an example quantitative assessment of miRNA inHepa1-6 and HeLa cell cultures before (low density) and at confluence(high density), according to an embodiment. The horizontal axis 822indicates a grouping of data; the vertical axis 824 indicates miRNAconcentration as a percent of total RNA in sample as measured usingselective ITP with gradients in one or more additives. Bar 832 and bar842 represent HeLa and Hepa1-6, respectively, at low density. Bar 834and bar 844 represent HeLa and Hepa1-6, respectively, at high density.The error bars 828 represent 95% errors.

For these graphs, miRNA quantitation was performed with ITP as describedabove. Absolute miRNA abundance from total RNA was measured fromsubconfluent and confluent cultures. The results for HeLa and Hepa1-6cells in FIG. 8B are presented as percentage of total RNA (since totalRNA concentration does not vary with cell density). In both cases, asignificant increase was observed in miRNA expression between thesubconfluent and confluent cultures. miRNA levels increased from 0.11%to 0.32% of total RNA in HeLa cells, and from 0.24% to 0.42% in Hepa1-6cells. These results provide independent validation of the findingsreported in Hwang et al. and confirm the efficacy of the ITP based miRNAquantification. While relative values of miRNA levels were qualitativelysimilar to Hwang's study, it is noted that the current measurements showslightly larger concentrations of miRNA than levels estimated frommicroarray data. We attribute this apparent discrepancy to variations ofmiRNA expression between different cell types and to different small RNAextraction efficiencies associated with the preparation method.

4.3 Molecular Beacons for microRNA

Molecular beacons are sequence specific nucleic acid probes thatfluoresce upon hybridization. Developed in the early years ofquantitative PCR, molecular beacons have become ideal sequence-specificfluorescent reporters for nucleic acid amplifications assays and in vivohybridization. The sequence specific fluorescence of MBs originates fromtheir unique structure shown in FIG. 9A. FIG. 9A is a block diagram thatillustrates a sequence specific molecular beacon (MB), used in someembodiments. MBs are composed of four units: (i) a nucleic acid probesequence 942 (in a loop up to about 30 nt long) complementary to thetarget sequence of interest, e.g., on the analyte; this sequence isflanked by (ii) two, complementary self-hybridizing sequences in thestem 944 which allow conformation of the probe into a hairpin structure,(iii) a fluorophore 946 at the 5′ end, and (iv) a suitable quencher 948at the 3′ end. When the molecular beacon is free in solution, itacquires a hairpin structure which brings 5′ and 3′ ends to proximity,so the quencher hampers fluorescence. In the presence of a sequence 950complementary to the probe, the hairpin opens and hybridizes to thetarget sequence 950, e.g., as found in an analyte. This isthermodynamically favorable because the short stem hybrid 944 is lessstable than the longer probe-target hybrid. In this configuration, thedistance between fluorophore and quencher is sufficient to enablefluorescence.

Here is presented a different hybridization strategy than conventionallyused for miRNA detection and quantification, which leveragesisotachophoresis (ITP) and hybridization with molecular beacons (MBs)for the profiling of miRNA. In contrast to conventional methods, thisassay is a single, amplification-free process which simultaneouslypurifies, preconcentrates, actively mixes, hybridizes, and produces anoptical signal whose intensity increases with the initial target sampleconcentration. The technique of spatial gradient additives is used.Here, a three-stage ITP channel with varying concentrations of(electrically neutral) polymer sieving matrix and denaturant is alsoused. However, in the third zone, a labeled probe is used to label onlycertain sequences of miRNA through hybridization of the probe with thetarget miRNA. In some embodiments, the third zone includes one or moreadditives to encourage hybridization or label sensitivity. In theillustrated embodiment, magnesium ion in the channel is also introducedas an additive to promote hybridization. These three serial zonesallowed for consecutive pre-concentration, selection and hybridizationof miRNA. Thus, in some embodiments, the analyte is a nucleotide and afourth additive comprises a nucleotide probe with a fluorescent label.

FIG. 9B and FIG. 9B are block diagrams that illustrate pre-concentrationof the analyte in a first zone, separation of similar speed ions by sizeusing a higher concentration of one additive in a second zone, anddetection using hybridization with molecular beacons based on higherconcentration of another additive in a third zone, according to anembodiment. FIG. 9B depicts a preconcentration first zone 902, an miRNAand MB selection second zone 904 and a hybridization and detection thirdzone 906. Concentration of polymer 908 in solution is indicated by crosshatching in which wider separation indicates lower concentration of thepolymer in solution. The polymer concentration increases to a maximum inthe second zone 904. In the preconcentration zone 902 the first LEmixture (e.g., LE1 described above with reference to FIG. 2A) allowsmiRNA and RNA ions to focus at the interface 512 between TE and LE ions.In the miRNA selection zone 904 the second LE mixture (e.g., LE2described above with reference to FIG. 2A) allows miRNA in a leadingfocused interface 916 and causes RNA ions to de-focus in a followingpopulation peak 914, with denaturing agents enhancing the separation. Inthe hybridization and detection zone 906 the third LE mixture (e.g., LE3described above with reference to FIG. 2A) labels the miRNA in theleading focused interface 516 without hindrance by denaturing agents.

FIG. 9C is a block diagram that depicts TE ions 922, LE ions 924, miRNAions 926, miRNA target ions 928 that have the target sequence, molecularbeacon molecules 932 in the LE3 mixture that will bind only to the miRNAtarget ions, and hybridized target molecules 934 in the ITP focusedinterface of the third zone 906. In zone 906 the miRNA target ions 926take up a molecular beacon 932 and stay focused between the TE ions 922and the LE ions 924

Here, mature miRNA (18 to 24 nt) are selectively focused and all RNAmolecules longer than 60 nt rejected from the ITP zone. Therefore biasis avoided from hybridization of long RNAs that contain identical orsimilar sequences. In particular, the 70 nt long miRNA precursorspre-miRNA are excluded. This selectivity combined with the simultaneoushybridization is in some ways similar to the process of northernblotting, which requires multiple successive steps includingelectrophoresis and hybridization to achieve detection. In contrast tonorthern blotting, ITP here provides simultaneous preconcentration andactive mixing of target and probe prior to and during detection.

The sample is mixed in the TE which includes 5 mM MOPS, 5 mM Tris and92.5% v/v formamide. TE and sample are loaded into the input port 212 ofFIG. 2A. In the first zone mixture LE1, a low (0.5% w/v PVP) sievingmatrix polymer concentration and 7 M urea are included. The mobility ofmiRNA increases with decreasing polymer concentration, so LE1 yields astrong flux of RNA to the ITP interface, but molecules longer than(mature) miRNA molecules are also focused. Also, the slightly largerconcentration of leading ions in LE1 (50 mM) augments ITPpreconcentration dynamics. The second zone with mixture LE2 has largepolymer concentration (3% w/v PVP), which globally decreases mobility ofRNA. This defocuses longer RNA (they fall behind and out of the ITPfocus zone) while leaving miRNA and MB focused, at the cost of locallyretarding focusing dynamics. As discussed below, the cut off length isbelow 60 nt, so that miRNAs can focus, but pre-miRNAs cannot focus.Finally, mixture LE3 in the third zone has low denaturing conditions (2M urea, lower polymer concentration of 0.5% w/v), and optimizedmagnesium chloride concentration (2 mM Mg²⁺). These conditions enablefast hybridization, and optimize fluorescence signals as miRNA targetsspecifically bind to MBs.

Initially, MB probes targeting the miRNA of interest are dissolved inthe three LE mixtures, and total RNA (which includes miRNA) is dissolvedin the TE. Leading and trailing ions are selected so that theirmobilities allow for simultaneous and co-located focusing of miRNAincluding target miRNA, MB probe, and the miRNA-probe hybrid. (Thelatter ITP format which focuses multiple analytes into a common, sharpzone is called peak mode ITP.) Under this condition, miRNA and MB andthe hybrid simultaneously focus at the interface between TE and LE. Inthe laboratory frame, miRNA overspeed TE ions and other RNA, and migratetoward the ITP zone. At the same time, the ITP zone overtakes andfocuses MBs initially in the LE, so target and probe are activelypreconcentrated and driven into themigrating ITP interface. In thisfocused interface, and under optimized conditions, miRNA hybridizes tothe probe sequence of the MB, disrupting their hairpin structure andyielding a sequence-specific increase in fluorescence intensity withinthis ITP interface. This way, the focused interface acts as a reactorvolume defined by its axial width and the cross sectional area of themicrochannel. In the 44 micron wide, 12 micron deep channel of chip 200,the volume of the ITP reaction zone is on the order of 10 pL, givenobserved ˜10 micron wide ITP interfaces. This is a significantly smallerreaction volume compared to existing microfluidic reactors, which are atleast on the order of few nanoliters. The strong preconcentrationdynamics (on the order of 10³ to 10⁴ fold increase of reactants in theseconditions) yield improved kinetics and sensitivity.

Leading electrolytes contain DNase- and RNase-free Tris hydrochloridebuffer (pH=8.0) from Invitrogen of Carlsbad, Calif.,polyvinylpyrrolidone (PVP, M.W.=1,000,000) from Polysciences Inc. ofWarrington, Pa., urea from EMD biosciences of Gibbstown, N.J., andmagnesium chloride from EMD biosciences. Concentrations in mixtures LE1,LE2 and LE3 are respectively 50, 20 and 20 mM of Tris hydrochloride;0.5% w/v, 3% w/v and 0.5% w/v of PVP; 7, 7, and 2 M of urea; 0, 2 and 2mM of magnesium chloride. The TE is a solution of 5 mM Tris fromSigma-Aldrich of Saint Louis, Mo., and 5 mM MOPS from Sigma-Aldrich in92.5% v/v formamide UltraPure from Invitrogen. All solutions wereprepared with DNase- and RNase-free deionized water from Gibco ofCarlsbad, Calif.

HPLC-purified molecular beacons and synthetic miRNA were purchased fromIntegrated DNA Technologies of Coralville, Iowa DNA molecular beacons,5′-labeled with TYE 665 fluorescent dye (excitation at 645 nm andemission at 665 nm) and 3′-labeled with Iowa Black RQ quencher (peakabsorbance at 656 nm) were used. The precursor mir-26a was synthesizedand PAGE-purified by Dharmacon of Lafayette, Colo. The sequences ofsynthetic oligoribonucleotides and probes used in this work are listedin Table 2. For molecular beacons, TYE 665 is a fluorophore (withspectrum similar to Cy5) and Iowa Black RQ (IBRQ) is the quencher. ThemiRNAs and precursor are ribonucleic acids while molecular beacons aredeoxyribonucleic acids. For molecular beacons, the complementarystem-forming sequence fragments are underlined. Total RNA from normalhuman liver and kidney were obtained from as FirstChoice human total RNAfrom Ambion of Austin, Tex. Before each experiment, the sample (total orsynthetic RNA) was dissolved to the specified concentration in 50 μL ofTE, placed in a water bath at 70° C. for 5 min and finally on ice untilrunning the ITP hybridization experiment. Separately, the MB wasdissolved in 500 μL of each LE mixture.

TABLE 2 Nucleic acids used in molecular beacon experiments SEQ.Oligo name ID. Sequence  (length) NO (5′ to 3′) miR-26a  6UUCAAGUAAUCCAGGAUAGGCU (22 nt) miR-126  4 UCGUACCGUGAGUAAUAAUGCG (22 nt)miR-122  7 UGGAGUGUGACAAUGGUGUUUG (22 nt) mir-26a  8GUGGCCUCGUUCAAGUAAUCGGAUACA (77 nt) GGCUGUGCAGGUCCCAAUGGGCCUAUU (miR-26aCUUGGUUACUUGCACGGGGACGC precursor) miR-26a MB  9TYE665-CCGAGCAGCCTATCCTGGAT (34 nt) TACTTGAAGCTCGG-IBRQ miR-122 MB 10TYE665-CCGAGCCAAACACCATTGTC (34 nt) ACACTCCAGCTCGG-IBRQ

10A is a graph that illustrates example demonstration of the ITPhybridization assay compared to a control, according to an embodiment.The horizontal axis 1002 is time in seconds; and, the vertical axis 1004is fluorescence intensity in arbitrary units. Trace 1010 represents anegative control displaced +0.5 s and +0.7 A.U. on the plot for clarityof presentation. Trace 1012 represents a sample with target miRNA. Thetraces represent two isotachopherograms acquired 8 mm into the LE3 zone.In both experiments, the LE contains 1 nM of MB targeting miR-26a. Theupper trace 1010 corresponds to a negative control experiment where theTE contains no RNA. This trace exhibits a peak which is attributed toimperfect quenching of the focused MB. The trace 1012 shows the resultof ITP-hybridization where we added 1 nM of miR-26a target to the TE.The ITP peak has significantly greater amplitude compared to thenegative control. This demonstrates successful combination of ITP and MBbased hybridization for the detection of miRNA.

FIG. 10B is a graph 1020 that illustrates the area of each peak of thetraces in FIG. 10A, according to an embodiment. Horizontal axisindicates negative control 1022 and sample with target miRNA 1022 b. Thevertical axis represents area in the peak in arbitrary units. The peakarea of this experiment with 1 nM target in the TE (+) at 1022 b is morethan 6 times larger than the area of the negative control (−) at 1022 a.This shows successful hybridization of the target and MBs within thefocused zone.

For an ITP hybridization experiment with a peak area A, we define therelative fluorescence enhancement f as:f=A/A _(nc)−1,where A_(nc) is the peak area of the negative control, i.e. anexperiment with equal MB concentration but a blank TE. In the embodimentpresented in FIG. 10B, f is approximately 5.5. f theoretically variesbetween zero (when A=A_(nc)) and a saturation value where all focused MBare open. The latter occurs when the number of target copies in thefocused zone is much larger than the number of MBs. We note that f isalso a function of other parameters including MB concentration, MB stemsequence, miRNA melting temperature, and ITP chemistry.

The fluorescence enhancement of MBs increases with target concentration.In peak mode ITP, the amount of focused sample is a linear function ofsample concentration in the TE. Consequently, in the ITP hybridizationassay, f increases with target concentration in the TE. Titrationexperiments were performed to illustrate the effect of sampleconcentration on fluorescence enhancement. FIG. 11 is a graph thatillustrates typical example fluorescence enhancements f for ITPhybridization, according to an embodiment. The horizontal axis 1102 isRNA concentration in moles (M); and, the vertical axis is relativefluorescence enhancement f.

FIG. 11 reports fluorescence enhancements f for ITP hybridization at 100pM MB with miR-26a concentrations 1112 ranging from 1 pM to 100 nM inthe TE (circles). To aid in data visualization, the data was fitted withspline functions (dashed lines). Titration with miR-26a shows the signalgenerated from hybridization of the perfectly matching target.Fluorescence enhancement remains small at low concentration (below 10pM) and significantly increases at 100 pM and above. f plateaus overabout 10 nM, where nearly all focused MBs are open. Potential unspecifichybridization was also verified by titrating with miR-126 (whosesequence is distinct from miR-26a) plotted as trace 1114, and observedthat fluorescence enhancement remained approximately null at allconcentrations. This confirms the specificity of MB hybridization in theITP zone. Titration with the precursor large miR-26a sample indicated bytrace 1116 shows only slow increase of fluorescence with concentrationabove 10 nM, since the longer molecules are filtered out by the ITPprocess in the polymer zone. This shows that ITP in the LE2 zoneexcludes miRNA precursors from the focused zone, and allows forselective hybridization on miRNA. That is, this demonstrate that in ITPhybridization, MBs bind specifically to the correct target sequence.

To show the efficacy of the ITP hybridization assay in a biologicallyrelevant case, detection of miR-122 was performed in two human tissuetotal RNA samples. The following liver-specific miRNA target was chosenfor its large dynamic range of expression: miR-122 is highly expressedin liver but poorly expressed in other organs. We diluted total RNA fromhuman liver and kidney in TE down to 10 ng·μL⁻¹. We then performed theITP hybridization assay on these samples with 100 pM MBs in the LEtargeting miR-122. FIG. 12 is a graph that illustrates example resultsof the ITP hybridization assay for miRNA in liver tissue compared to acontrol, according to an embodiment. The horizontal axis 1202 is time inseconds and the vertical axis 1204 is fluorescence intensity inarbitrary units. Trace 1212 indicates the results for the sample withliver cells and trace 1214 shows the results for a negative control. Inboth experiments, the LE contains 100 pM of MB targeting miR-122. Trace1214 corresponds to a negative control where the TE contains no RNA.Trace 1212, displaced up and to the right for clarity of presentation,shows the result of ITP-hybridization where we added 10 ng·μL−1 of humanliver total RNA to the TE. The ITP peak has significantly greateramplitude compared to the negative control.

FIG. 13A is a graph that illustrates example demonstration of ITPhybridization assay for detection and quantification of miR-122 inkidney and liver, according to an embodiment. The horizontal axisindicates three groups of results, for negative control 1302 a, kidneyRNA 1302 b and liver RNA 1302 c. The vertical axis 1324 indicatesenhancement f Peak areas of ITP hybridization experiments are plottedwhere LEs initially contain 100 pM MB s targeting miR-122. Theexperiments shown have TEs which contain: a blank (left bar 1302 a), 10ng·μL⁻¹ of total RNA from human kidney (middle bar 1302 b), and 10ng·μL⁻¹ of total RNA from human liver (right bar 1302 c). The increasein fluorescence for kidney over the control is not statisticallysignificant, showing our assay predicts miR-122 concentration in kidneybelow a limit of detection of 3,000 copies per cell. The peak area forliver is significantly greater, indicating greater expression ofmiR-122. Uncertainty bars 1303 represent 95% confidence on the mean.

A calibration curve built using synthetic miR-122 is used to estimatetarget concentration from fluorescence enhancement. FIG. 13B is a graphthat illustrates an example calibration curve resulting frominterpolation of hybridization results from synthetic miR-122 as afunction of concentration, according to an embodiment. The solid lineshows a calibration curve resulting from interpolation of hybridizationresults from synthetic miR-122 versus concentration (“x” symbols 1330).This curve is used to calculate the concentration corresponding to theenhancement f_(liver)=1.3. This concentration estimate is 10.3 pM,corresponding to approximately 16,000±400 copies per cell. Uncertaintybars represent 95% confidence on the mean.

These experimental embodiments showed that ITP hybridization enableslength-selective detection of miRNA and can distinguish miRNA from itsprecursors. They also showed that the sequence specificity of MBs wasunaffected by coupling hybridization with ITP. Furthermore, theydemonstrated the efficacy of the assay for the detection of miRNAtargets in total RNA. They successfully detected miR-122 in liver andcorroborated reduced expression in kidney. Using calibrationexperiments, the amount of miR-122 in liver was calculated; and theestimate is in fair agreement with measurements performed with otherquantification methods.

4.4 Use of Spacer Ions

In some embodiments, gel sieving matrix concentration gradients are usedin ITP to first enhance reaction kinetics and then separate reactants indistinct focused zones, as shown in FIG. 14A. A spacer compound is addedwith mobility higher than that of the formed reactants but lower thanthat of the unreacted compounds within the gel matrix. As describedabove with reference to FIG. 4B, UV light is used to create a discretegel region within a microchannel, thus partitioning the channellongitudinally into three separate regions. Molecular beacons and theircomplementary targets are used as example analytes. All analytes focusinitially in free solution in peak mode at the spacer-LE interface. Uponentering the discrete gel region, the beacon-target complex mobility isreduced to below that of the spacer but not below that of the TE. Thebeacon mobility, however, remains bracketed by the LE and spacer. Thisenables separation of excess beacons from beacon-target hybrid, followedby refocusing of the beacon-target hybrid at the TE-spacer interface.Separation of excess beacon from beacon-target hybrid increases assaysensitivity by reducing the signal from imperfect quenching observed inother embodiments. This technique is demonstrated by enhancing thereaction between molecular beacons and a complementary 77nt precursormicro-RNA target in an 8% polyacrylamide gel matrix. The oligo sequencesare given in Table 2. For this demonstration, miR-26a precursor was usedas the target and miR-26a MB was used as the molecular beacon.

FIG. 14A is a block diagram that illustrates pre-concentration of theanalyte and reporter ahead of a spacer ion in a first zone, andseparation of bound and unbound reporter ions by the spacer ions using ahigher concentration of one additive in a second zone, according to anembodiment. FIG. 14B depicts LE ions 1422, TE ions 1424, spacer ions1426, target DNA ions 1432, molecular beacon ions 1434 and MB-targethybrid ions 1436, in each of the two zones 1402 and 1406. The TE spacerand target mixture at input port 1442 is at electrical ground 234 andthe LE and MB mixture at output port 1444 is at a high voltage 236. Inzone 1402 the target, spacer and MB begin to focus between the LE andTE. The MB, and target and hybrid product all have higher mobility thanthe spacer ion and thus focus between the spacer and LE as shown in thegraph below with horizontal axis 1452 indicating distance along thechannel and vertical axis 1454 indicating electrical field strength. TheMB, target and MB-target hybrid are all focused at 1456, ahead of thespacer and behind the LE. In zone 1406, where the focus interfaceencounters the gel, the MB-target hybrid ions suffer reduced mobilityand below that of the spacer ion and above that of the TE. The unboundtarget and MB, which are each half the size of the hybrid, retainmobilities greater than the spacer ion. This is reflected in the graphbelow with horizontal axis 1462 indicating distance along channel,vertical axis indicating electric field strength. The unbound target andMB are focused in interface 1466 between the spacer ions and LE. TheMB-target hybrids are focused in the interface 1467 between the spacerions and the TE.

This result is brought about by the following steps. During step 305,the channels are filled with a buffer that contains the leadingelectrolyte (LE), acrylamide/bisacrylamide monomer and cross-linker, anda photo-initiator. In this experiment the following were used: 50 mMHydrochloric Acid (HCl) as LE, 100 mM Tris as a counte ion, 6% (w/v)acrylamide as monomer, 3.3% (w/w) bisacrylamide as cross-linker, and0.2% (w/v) VA-086 as a photo-initiator. A mask is used to cover regionsof the channel, allowing spatially-selective exposure to UV light, thusphoto-patterning desired regions with high resolution. During step 311,a buffer containing the trailing electrolyte (TE), a spacer compound,and the analytes (in this case, a molecular beacon and its complementarytarget) is added to one of the wells, replacing the LE. SEQ. ID. NO 8was used as synthetic oligoribonucleotides and SEQ. ID. NO. 9 was usedas molecular beacon probes. During step 313, an electric field isapplied using electrodes in the wells. ITP is thus carried out, focusingthe analytes between the fast LE ions and the slow TE ions. Themolecular beacon and its target will react, forming a complex, causingthe stem-loop structure of the beacon to loosen and open, thusseparating the fluorophore from its quencher, and increasing thefluorescence by nearly an order of magnitude. Free beacon, target, andbeacon/target hybrid focus together at the spacer-LE interface. As thereactants enter the sieving matrix, the retarding effects of the gelreduce the mobility of the larger beacon-target complex below that ofspacer ions. Unreacted beacons remain focused ahead of the spacercompound and beacon/target hybrid re-focuses at the TE-spacer interface.

FIG. 14B is a graph that illustrates example measurements of bound andunbound reporter ions in the arrangements of FIG. 14A, according to anembodiment. The horizontal axis 1472 is distance along the channel inmm. The vertical axis 1474 is fluorescence intensity in arbitrary units.In this experimental embodiment the target is a 77nt precursormicro-RNA. In the absence of target molecules trace 1490 results,displaced vertically about 6 units for clarity of presentation, showsmolecular beacons focus primarily in one peak. The signal intensity bandbelow trace 1490 shows weak fluorescent signal intensity due to theimperfect quenching of the fluorophore, and represents baselineintensity. In the presence of 1 μM precursor micro-RNA molecules trace1480 results. Trace 1480 includes two peaks in the gel region, separatedby the spacer ions, the latter peak being of significantly higherintensity, indicating the opening of the beacon, distancing thefluorophore from its quencher. The signal intensity band 1488 belowtrace 1480 shows strong fluorescent signal intensity at the latter peak.Inset images are taken from experiment visualizations.

4.5 Use of Reporter-Binding Agent Fixed in Polymer Gel

Spatial gradients of a modified gel capture matrix are used to firstreact target molecules and fluorescent reporters in ITP and thenimmobilize excess reporters, as shown in FIG. 15. This enables detectionof target molecules, which remain bound to reporters. Anacrylamide-modified probe is included with the monomer solution. Thisprobe becomes incorporated into the polymers upon exposure to UV lightand remains immobilized in the gel matrix. A discrete gel regioncontaining capture probe is created within a microchannel, thuspartitioning the channel axially into three regions.

FIG. 15A is a block diagram that illustrates pre-concentration of theanalyte and reporter, separation of bound and unbound reporter ionsusing a higher concentration of polymer sieve that fixes areporter-binding probe in a second zone, and third zone of onlyanalyte-bound reporter ions in a third zone with no probe fixed in apolymer sieve, according to an embodiment. In the first zone 1502,target molecules are focused at interface 1512 and react withcomplementary reporter probes end-labeled with a fluorophore. In thesecond, gel-filled zone 1504, excess reporter probes become immobilizedto the gel matrix 1508 via interaction (e.g. hybridization) withacrylamide-modified probes immobilized in the gel. Reporter probes boundto a target molecule remain focused at the ITP interface 1514 throughoutthis region as a result of slow off-rates. This enables detection oftarget molecules through visualization of the fluorescent reporter inthe interface 1516 of the third zone 1506.

This technique is demonstrated by detecting target miR-15a molecules,using a complementary oligonucleotide capture probe modified withAcrydite, in a 4% polyacrylamide gel. The LE buffer is composed of 100mM HCl, 200 mM Tris, and 2.5 mM MgCl. The channels are filled with LEbuffer 1520, containing 10 μM acrylamide-modified (commercial nameAcrydite-modified), 4% T 3% C acrylamide/bisacrylamide monomer andcrosslinker 1% PVP (MW 1,000,000), and 0.13% VA-086 photoinitiator. Amask is used to photo-pattern desired regions of the channel. Electricfield is applied using electrodes in the wells to remove unincorporatedcapture probe away from the capture region. A TE buffer 1522 containinga fluorescent reporter and target molecules is added to one of thewells, replacing the LE. The sequences of synthetic oligoribonucleotidesand probes used in this work are listed in Table 3. Electric field isapplied using electrodes in the wells. ITP is thus carried out, focusingthe analytes between the fast LE ions and the slow TE ions. The reporterprobes and complementary target molecules initially react in freesolution, forming a target/reporter hybrid. As the ITP interface entersthe gel region, excess reporters hybridize with the immobilizedcomplementary oligonucleotides and therefore become immobilized in thegel matrix. Due to low off-rates, the target/reporter complex remainshybridized throughout the gel region. The fluorescent signal is measuredin the third (free solution) region. Signal intensity is proportional toinitial concentration of target molecules.

TABLE 3 Nucleic acids used in reporter-binding agent experimentsOligo name SEQ. (length) ID. NO Sequence (5′ to 3′) miR-15a 11UAGCAGCACAUAAUGGUUUGUG (22 nt) miR-15a 12 Acrydite-GTAGCAGCACATAAProbe (23 nt) TGGTTTGTG miR-15a Reporter 13 Cy3-CACAAACCATTATGT (23 nt)GCTGCTA

FIG. 15B is a graph that illustrates example measurements ofanalyte-bound control in the arrangements of FIG. 15A, according to anembodiment. The horizontal axis 1532 is distance along the channel atthe viewing point. The vertical axis 1534 is fluorescence signalintensity in arbitrary units. In the detection of miR-15a, a 10 μMacrylamide-modified oligonucleotides and 10 nM fluorescent reporter areused. In the presence of zero target molecules indicated by trace 1540and image 1542, reporter probes become immobilized in the gel capturematrix. This results in a weak fluorescence signal. In the presence of10 nM target molecules (miR-15a) indicated by trace 1550 and image 1552,fluorescent reporters hybridize to target molecules and thus remainfocused in ITP. This results in a strong fluorescence signal.

5. Alternative Embodiments

In alternative embodiments, one or more analytes, reporters or productmolecules include one or more of the sequences described in thissection.

It is known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively). The terms “start codon region” and “translationinitiation codon region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationcodon region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation termination codon.

The open reading frame (ORF) or “coding region,” is known in the art torefer to the region between the translation initiation codon and thetranslation termination codon. It is also known in the art that variantscan be produced through the use of alternative signals to start or stoptranscription and that pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites.

In the context of various embodiments, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anucleic acid is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the nucleic acid and theDNA or RNA are considered to be complementary to each other at thatposition. The nucleic acid and the DNA or RNA are complementary to eachother when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides that can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termsthat are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between thenucleic acid and the DNA or RNA target.

Various conditions of stringency can be used for hybridization as isdescribed below. As used herein, the term “hybridizes under lowstringency, medium stringency, high stringency, or very high stringencyconditions” describes conditions for hybridization and washing. Guidancefor performing hybridization reactions can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6, whichis incorporated by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: 1) lowstringency hybridization conditions in 6.times.sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by two washes in 0.2.times.SSC,0.1% SDS at least at 50.degree C. (the temperature of the washes can beincreased to 55° C. for low stringency conditions); 2) medium stringencyhybridization conditions in 6.times.SSC at about 45° C., followed by oneor more washes in 0.2.times.SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions in 6.times.SSC at about 45° C., followed by oneor more washes in 0.2.times.SSC, 0.1% SDS at 65° C.; and preferably 4)very high stringency hybridization conditions are 0.5M sodium phosphate,7% SDS at 65° C., followed by one or more washes at 0.2.times.SSC, 1%SDS at 65° C. Very high stringency conditions (4) are the preferredconditions and the ones that should be used unless otherwise specified.

Nucleic acids in the context of various embodiments include“oligonucleotides,” which refers to an oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. DNA/RNA chimeras are alsoincluded.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure; however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Oligonucleotides containing modified backbones or non-naturalinternucleoside linkages can be used. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides. Preferred modified oligonucleotidebackbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonatesincluding 3-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be a basic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference. Preferredmodified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

In some oligonucleotide mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,an oligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments of some embodiments use oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂).sub.nNH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂).sub.nCH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

A further modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring thereby forming a bicyclic sugar moiety. The linkage is preferablya methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226.

Other modifications include 2′-methoxy(2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro(2′-F). The 2′-modification may be in thearabino (up) position or ribo (down) position. A preferred 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotidesand the 5′ position of 5′ terminal nucleotide. Oligonucleotides may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative United States patents that teachthe preparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine. (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C.ident.C—CH₃) uracil andcytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified nucleobases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof some embodiments. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Another modification of the oligonucleotides for use in some embodimentsinvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of someembodiments can include conjugate groups covalently bound to functionalgroups such as primary or secondary hydroxyl groups. Conjugate groups ofsome embodiments include intercalators, reporter molecules, polyamines,polyamides, poly ethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of various embodiments, include groups that improveoligomer uptake, enhance oligomer resistance to degradation, and/orstrengthen sequence-specific hybridization with RNA. Groups that enhancethe pharmacokinetic properties, in the context of various embodiments,include groups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et. al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid;e.g., di hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of some embodiments mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. “Chimeric” compounds or“chimeras,” in the context of various embodiments, are oligonucleotides,which contain two or more chemically distinct regions, each made up ofat least one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids.

The oligonucleotides used in accordance with various embodiments may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed.

In the foregoing specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the invention. Thespecification and drawings are, accordingly, to be regarded in anillustrative rather than a restrictive sense.

What is claimed is:
 1. An isotachophoresis method comprising: (a)forming a concentration gradient of a matrix material along a channel,wherein the channel connects an input port to an output port, andwherein the input port is configured to receive a sample comprising ananalyte; (b) introducing ions into the channel, wherein the ionscomprise (i) a leading electrolyte having a first effective mobilitymagnitude greater than an effective mobility magnitude of the analyte,and (ii) a trailing electrolyte having a second effective mobilitymagnitude less than the effective mobility magnitude of the analyte; (c)contacting the analyte to the leading electrolyte; (d) contacting thetrailing electrolyte to the analyte; and (e) applying an electric fieldto the channel, wherein the matrix material affects at least one of theanalyte, the leading electrolyte or the trailing electrolyte in at leasta portion of the channel.
 2. The method of claim 1, wherein the matrixmaterial comprises a stationary material.
 3. The method of claim 2,wherein the matrix material comprises a stationary polymeric material.4. The method of claim 1, wherein the matrix material is ion permeable.5. The method of claim 1, further comprising detecting the analyte. 6.The method of claim 1, wherein the analyte is a protein.
 7. The methodof claim 1, wherein the analyte is a nucleic acid.
 8. The method ofclaim 1, further comprising introducing an additional ion into thechannel via an additional channel at a junction, wherein the additionalion is different from the leading electrolye, the trailing electrolye,and the analyte.
 9. The method of claim 8, wherein prior to theintroducing the additional ion, the additional ion is substantiallyabsent from the channel.
 10. The method of claim 8, wherein theadditional ion comprises a weak base or a weak acid.
 11. The method ofclaim 8, wherein the introducing the additional ion is conducted afterthe analyte has migrated along the channel at least to the junction. 12.The method of claim 8, wherein the introducing the additional ion istriggered by a feedback signal.
 13. The method of claim 12, wherein thefeedback signal is triggered by detection of an optical signal or anelectrical signal.
 14. An apparatus comprising: (a) a channel connectingan input port to an output port, wherein the input port is configured toreceive a sample comprising an analyte; (b) a concentration gradient ofa matrix material located in the channel; (c) a leading electrolyte,wherein the leading electrolyte has a first effective mobility magnitudegreater than an effective mobility magnitude of the analyte; (d) atrailing electrolyte, wherein the trailing electrolyte has a secondeffective mobility magnitude less than the effective mobility magnitudeof the analyte; and (e) an electric field generator capable of applyingan electric field to the channel, wherein the matrix material affects atleast one of the analyte, the leading electrolyte or the trailingelectrolyte in at least a portion of the channel.
 15. The apparatus ofclaim 14, wherein the matrix material comprises a stationary material.16. The apparatus of claim 15, wherein the matrix material comprises astationary polymeric material.
 17. The apparatus of claim 14, whereinthe matrix material is ion permeable.
 18. The apparatus of claim 14,wherein the matrix material is functionalized with at least one type ofcapture molecule.
 19. The apparatus of claim 14, wherein the apparatusfurther comprises a detector.
 20. The apparatus of claim 14, wherein theanalyte is a protein.
 21. The apparatus of claim 14, wherein the analyteis a nucleic acid.
 22. The apparatus of claim 14, further comprising anadditional channel connected to the channel.
 23. The apparatus of claim22, further comprising an additional ion in the additional channel. 24.The apparatus of claim 22, wherein the additional channel comprises aside channel connected to the channel at a junction and wherein theelectric field generator is configured to apply an electric field to theside channel when the analyte migrates through the channel to thejunction.
 25. The apparatus of claim 23, wherein the additional ioncomprises a weak base or a weak acid.
 26. The apparatus of claim 23,wherein the additional ion is not present in the channel.
 27. Theapparatus of claim 24, wherein the application of the electric field tothe side channel is activated by a feedback signal.
 28. The apparatus ofclaim 24, wherein the feedback signal is triggered by detection of anoptical signal or an electrical signal.
 29. An isotachophoresis methodcomprising: (a) forming a concentration gradient of a matrix materialalong a channel, wherein the channel connects an input port to an outputport, and wherein the input port is configured to receive a samplecomprising an analyte; (b) introducing ions into the channel, whereinthe ions comprise (i) a leading electrolyte having a first effectivemobility magnitude greater than an effective mobility magnitude of theanalyte, and (ii) a trailing electrolyte having a second effectivemobility magnitude less than the effective mobility magnitude of theanalyte; (c) contacting the analyte to the leading electrolyte; (d)contacting the trailing electrolyte to the analyte; and (e) applying anelectric field to the channel, wherein the matrix material isfunctionalized with specific capture molecules.
 30. The method of claim29, wherein the specific capture molecules are present in aconcentration gradient along the channel.
 31. The method of claim 29,wherein the specific capture molecules comprise at least one of thegroup consisting of a macromolecule, a nucleic acid, an antibody, anantigen, biotin, avidin, a ligand, a receptor, a bait molecule, and aprotein.
 32. An isotachophoresis method comprising: (a) forming aconcentration gradient of a matrix material along a channel, wherein thechannel connects an input port to an output port, and wherein the inputport is configured to receive a sample comprising an analyte; (b)introducing ions into the channel, wherein the ions comprise (i) aleading electrolyte having a first effective mobility magnitude greaterthan an effective mobility magnitude of the analyte, and (ii) a trailingelectrolyte having a second effective mobility magnitude less than theeffective mobility magnitude of the analyte; (c) contacting the analyteto the leading electrolyte; and (d) contacting the trailing electrolyteto the analyte; and (e) applying an electric field to the channel,wherein the matrix material is functionalized with probe molecules.